Aptamer-functionalized silver nanoparticle pre-analytical enrichment integrated with loop-mediated isothermal amplification for improved detection of pathogenic Leptospira interrogans.
Abstract
Early diagnosis of leptospirosis remains challenging because the low and transient leptospiraemia during the acute phase often results in bacterial DNA concentrations below the analytical detection limit of conventional molecular assays. This proof-of-concept study evaluated the feasibility of integrating aptamer-functionalised silver nanoparticles (AgNPs) as a pre-analytical enrichment step with loop-mediated isothermal amplification (LAMP) to improve the molecular detection of pathogenic Leptospira interrogans. Unlike previously reported aptamer-nanoparticle systems that primarily function as biosensors, the proposed approach employs aptamer-functionalised AgNPs as an upstream enrichment platform prior to nucleic acid amplification. Silver nanoparticles were synthesised by sodium borohydride reduction and characterised using UV-visible spectroscopy, Fourier-transform infrared spectroscopy (FTIR), X-ray diffraction (XRD) and transmission electron microscopy (TEM), while aptamer conjugation was supported by a characteristic surface plasmon resonance shift from 390 to 404 nm. Compared with unfunctionalised AgNPs and unenriched controls, aptamer-functionalised AgNP enrichment resulted in significantly greater DNA recovery (21.6% versus 7.3% at 103 CFU/mL; p = 0.018). Enrichment-assisted LAMP achieved an analytical limit of detection of 10 CFU/mL, representing a 100-fold improvement over conventional LAMP (103 CFU/mL), and demonstrated improved amplification performance in plasma samples containing low bacterial concentrations. Preliminary evaluation using 52 clinically suspected canine samples yielded a diagnostic sensitivity of 88.5% and specificity of 100%. Although additional mechanistic studies, comprehensive nanoparticle characterisation and larger multicentre clinical validation are required, these findings demonstrate the feasibility of aptamer-functionalised AgNP enrichment as a promising pre-analytical strategy for improving molecular detection of low-abundance Leptospira in resource-limited laboratory settings.